Development of Semisynthetic Apoptosis-Inducing Agents Based on Natural Phenolic Acids Scaffold: Design, Synthesis and In-Vitro Biological Evaluation

A crucial target in drug research is magnifying efficacy and decreasing toxicity. Therefore, using natural active constituents as precursors will enhance both safety and biological activities. Despite having many pharmacological activities, caffeic and ferulic acids showed limited clinical usage due to their poor bioavailability and fast elimination. Therefore, semisynthetic compounds from these two acids were prepared and screened as anticancer agents. In this study, CA and FA showed very potent anticancer activity against Caco-2 cells. Consequently, eighteen derivatives were tested against the same cell line. Four potent candidates were selected for determination of the selectivity index, where compound 10 revealed a high safety margin. Compound 10 represented a new scaffold and showed significant cytotoxic activity against Caco-2. Cell-cycle analysis and evaluation of apoptosis showed that derivatives 10, 7, 11, 15 and 14 showed the highest proportion of cells in a late apoptotic stage.


Introduction
Cancer has the highest social and economic burden among all human diseases, and coupled with that, in the coming four decades it is anticipated to leap from the second to the first place to become the leading cause of death worldwide [1].
Over the years, several natural anticancer molecules were used as scaffolds for the synthesis of anticancer drugs, such as vinblastine and vincristine from Catharanthus alkaloids [2], paclitaxel from Yew trees [3], and podophyllotoxin from Podophyllum resins [4].
Ferulic acid has been used in healthy food and nutrition restoratives due to its wellknown antioxidant properties, and due to this biological effect, it can be used in Alzheimer's disease [25,26]. It is highly recommended for diabetic patients [27]. Moreover, FA has a reported neuroprotective effect [28][29][30] and relieves ischemia-reperfusion-induced cell apoptosis [31,32]. FA also has a chemo-preventive activity against carcinogenesis induced by 7,12-dimethylbenz[a]anthracene (DMBA) in rats [33,34]. Equally important is its antiproliferative action on osteosarcoma cells where it induces apoptosis by blocking the PI3K/Akt pathway [35].
It is highly recommended for diabetic patients as it can alleviate oxidative stress and also has an antihyperglycemic effect. [27] Recently, several phenolic compounds were tested against a spectrum of severe pathological conditions and both CA and FA showed potential results against coronavirus-based infections [36].
The aforementioned biological activities are achieved through a variety of mechanisms; the anticancer activity of both CA and FA is exerted by two mechanisms, acting as primary antioxidants that eliminate reactive oxygen species (ROS) and as secondary antioxidants (pro-oxidants) preventing the splitting of peroxides by metal ion chelation (iron and copper) [37,38]. Thus, inhibiting oxidative degradation of essential biological molecules (lipids, amino acids, polyunsaturated fatty acids and DNA bases) and preventing the formation of cellular lesions [25,[39][40][41][42].
As antioxidants, the study of the structure activity relationship of both acids and their derivatives showed that the hydroxy groups in caffeic and ferulic acids proved to be a pre-requisite for anti-oxidant activity, through their free radical scavenging and metal chelation properties. The 4-hydroxy moiety supports the resonance around the whole molecule resulting in higher stability due to the formation of the phenoxy radical. It is also accepted that a second 3-hydroxy substitution in caffeic acid promotes the antioxidant quality due to the formation of a stable ortho-quinone structure. Equally, in ferulic acid, the methoxy group enhances the resonance of the aromatic ring due to its electron-donating attributes. The acids have an additional aspect where the COOH tail provides lipid support and resonates with its neighboring unsaturation, offering alternative sites to prevent membrane deterioration by free radicals while the inclusion of H-donating groups in the side chain such as amide moiety accelerates the activity [43][44][45] (Figure 2). Caffeic acid and its different derivatives were reported to possess many biological activities such as immunostimulants [9][10][11][12], antibacterial [13,14], anti-inflammatory [15], cardio protective [15,16], anti-atherosclerotic [9,17], antidiabetic [15,18], antiproliferative [19,20], hepatoprotective [21], anticancer [9,[13][14][15][22][23][24], and Alzheimer's disease [25].
Ferulic acid has been used in healthy food and nutrition restoratives due to its wellknown antioxidant properties, and due to this biological effect, it can be used in Alzheimer's disease [25,26]. It is highly recommended for diabetic patients [27]. Moreover, FA has a reported neuroprotective effect [28][29][30] and relieves ischemia-reperfusion-induced cell apoptosis [31,32]. FA also has a chemo-preventive activity against carcinogenesis induced by 7,12-dimethylbenz[a]anthracene (DMBA) in rats [33,34]. Equally important is its antiproliferative action on osteosarcoma cells where it induces apoptosis by blocking the PI3K/Akt pathway [35].
It is highly recommended for diabetic patients as it can alleviate oxidative stress and also has an antihyperglycemic effect. [27] Recently, several phenolic compounds were tested against a spectrum of severe pathological conditions and both CA and FA showed potential results against coronavirus-based infections [36].
The aforementioned biological activities are achieved through a variety of mechanisms; the anticancer activity of both CA and FA is exerted by two mechanisms, acting as primary antioxidants that eliminate reactive oxygen species (ROS) and as secondary antioxidants (pro-oxidants) preventing the splitting of peroxides by metal ion chelation (iron and copper) [37,38]. Thus, inhibiting oxidative degradation of essential biological molecules (lipids, amino acids, polyunsaturated fatty acids and DNA bases) and preventing the formation of cellular lesions [25,[39][40][41][42].
As antioxidants, the study of the structure activity relationship of both acids and their derivatives showed that the hydroxy groups in caffeic and ferulic acids proved to be a prerequisite for anti-oxidant activity, through their free radical scavenging and metal chelation properties. The 4-hydroxy moiety supports the resonance around the whole molecule resulting in higher stability due to the formation of the phenoxy radical. It is also accepted that a second 3-hydroxy substitution in caffeic acid promotes the antioxidant quality due to the formation of a stable ortho-quinone structure. Equally, in ferulic acid, the methoxy group enhances the resonance of the aromatic ring due to its electron-donating attributes. The acids have an additional aspect where the COOH tail provides lipid support and resonates with its neighboring unsaturation, offering alternative sites to prevent membrane deterioration by free radicals while the inclusion of H-donating groups in the side chain such as amide moiety accelerates the activity [43][44][45] (Figure 2).
As cell apoptosis promoters, it was established that CA and FA play a distinctive role in encouraging cell apoptosis in human cervical cancer cells through inhibition of the Bcl-2 activity [46,47] and in non-small lung cancer cells (NSCLC) through the MAPK pathway [48] In another pathway, it was observed that the acids are responsible for suppressing hepatocellular carcinoma cells (HCC) via inhibiting the expression of vascular endothelial growth factor (VEGF), which in turn impedes angiogenesis of the tumor cells [49]. Molecules 2022, 27, x FOR PEER REVIEW 3 of 20 Figure 2. SAR of caffeic acid and ferulic acid as antioxidants.
As cell apoptosis promoters, it was established that CA and FA play a distinctive role in encouraging cell apoptosis in human cervical cancer cells through inhibition of the Bcl-2 activity [46,47] and in non-small lung cancer cells (NSCLC) through the MAPK pathway [48] In another pathway, it was observed that the acids are responsible for suppressing hepatocellular carcinoma cells (HCC) via inhibiting the expression of vascular endothelial growth factor (VEGF), which in turn impedes angiogenesis of the tumor cells [49].
It is worth mentioning that both CA and FA are highly selective, imparting cytotoxicity to tumor cells but not to normal cells; on the other hand, they are highly metabolized in the body, therefore jeopardizing their stability. By evaluating the two major derivatives-amides and esters-the amides possessed potent cytotoxic activity in addition to high in-vivo stability when put in comparison with the ester derivatives. Following in the same vein, 18 amide compounds (Table 1)  Therefore, the current study was conducted to verify the anti-tumor activity of CA and FA versus their semisynthetic derivatives on different cell lines and explore the underlying mechanisms using the following scheme:   It is worth mentioning that both CA and FA are highly selective, imparting cytotoxicity to tumor cells but not to normal cells; on the other hand, they are highly metabolized in the body, therefore jeopardizing their stability. By evaluating the two major derivatives-amides and esters-the amides possessed potent cytotoxic activity in addition to high in-vivo stability when put in comparison with the ester derivatives. Following in the same vein, 18 amide compounds (Table 1)

H
As cell apoptosis promoters, it was established that CA and FA play a distinctive role in encouraging cell apoptosis in human cervical cancer cells through inhibition of the Bcl-2 activity [46,47] and in non-small lung cancer cells (NSCLC) through the MAPK pathway [48] In another pathway, it was observed that the acids are responsible for suppressing hepatocellular carcinoma cells (HCC) via inhibiting the expression of vascular endothelial growth factor (VEGF), which in turn impedes angiogenesis of the tumor cells [49].
It is worth mentioning that both CA and FA are highly selective, imparting cytotoxicity to tumor cells but not to normal cells; on the other hand, they are highly metabolized in the body, therefore jeopardizing their stability. By evaluating the two major derivatives-amides and esters-the amides possessed potent cytotoxic activity in addition to high in-vivo stability when put in comparison with the ester derivatives. Following in the same vein, 18 amide compounds (Table 1) were synthesized ( Figure 3) via reacting the respective phenolic acids with a panel of amines, adopting N,N'-dicyclohexylcarbodiimide (DCC) as a coupling agent. Therefore, the current study was conducted to verify the anti-tumor activity of CA and FA versus their semisynthetic derivatives on different cell lines and explore the underlying mechanisms using the following scheme:  As cell apoptosis promoters, it was established that CA and FA play a distinctive role in encouraging cell apoptosis in human cervical cancer cells through inhibition of the Bcl-2 activity [46,47] and in non-small lung cancer cells (NSCLC) through the MAPK pathway [48] In another pathway, it was observed that the acids are responsible for suppressing hepatocellular carcinoma cells (HCC) via inhibiting the expression of vascular endothelial growth factor (VEGF), which in turn impedes angiogenesis of the tumor cells [49].
It is worth mentioning that both CA and FA are highly selective, imparting cytotoxicity to tumor cells but not to normal cells; on the other hand, they are highly metabolized in the body, therefore jeopardizing their stability. By evaluating the two major derivatives-amides and esters-the amides possessed potent cytotoxic activity in addition to high in-vivo stability when put in comparison with the ester derivatives. Following in the same vein, 18 amide compounds (Table 1) were synthesized ( Figure 3) via reacting the respective phenolic acids with a panel of amines, adopting N,N'-dicyclohexylcarbodiimide (DCC) as a coupling agent.
Therefore, the current study was conducted to verify the anti-tumor activity of CA and FA versus their semisynthetic derivatives on different cell lines and explore the underlying mechanisms using the following scheme:

Chemistry
The synthesis of CA and FA derivatives 3-20 is displayed in Figure 3. Preparation of these candidates was carried out by reacting the naturally obtained caffeic or ferulic acids with different amines using N,N'-dicyclohexylcarbodiimide (DCC) as a coupling agent for the preparation of the amide linkage starting from the acids. Separated amides 3-20 were purified by column chromatography. The main idea is screening the effect of changing the alkyl/cycloalkyl/aralkyl/aryl/heterocyclic moiety of the amide functionality on the biological activity. Compounds 1 and 2 exhibited a sky-blue color in UV, which turns to a greenish yellow color on exposure to ammonia vapor and spraying with AlCl3, indicating that they are phenolic acid in nature. 1 H NMR spectrum of 1 showed the characteristic signals for caffeic acid [50], which was characterized by two trans-olefinic protons at δ 6.16 and δ 7.41 ppm with a large coupling constant δ 15.88 Hz, assigned to H-8 and H-7, respectively. An ABX system was displayed at δ 6.76, 6.95 and δ 7.04 ppm assigned to H-5, H-6 and H-2, respectively. The 13 C NMR showed the nine characteristic carbon signals for 3,4-dihydroxy trans cinnamic acid with two oxy carbons at δ 146.0 and δ 148.5 (C-3 and 4), one carbonyl carbon at δ 168.3 (C-9), two trans olefinic carbons at δ 145.07 and δ 115.5 ppm, in addition to C-1 at δ 126.1, C-2 at δ 115.1, C-5 at δ 116.2 and C-6 at δ 21.6. 1 H NMR in encouraging cell apoptosis in human cervical cancer cells through inhibition of the Bcl-2 activity [46,47] and in non-small lung cancer cells (NSCLC) through the MAPK pathway [48] In another pathway, it was observed that the acids are responsible for suppressing hepatocellular carcinoma cells (HCC) via inhibiting the expression of vascular endothelia growth factor (VEGF), which in turn impedes angiogenesis of the tumor cells [49].
It is worth mentioning that both CA and FA are highly selective, imparting cytotoxicity to tumor cells but not to normal cells; on the other hand, they are highly metabolized in the body, therefore jeopardizing their stability. By evaluating the two major derivatives-amides and esters-the amides possessed potent cytotoxic activity in addition to high in-vivo stability when put in comparison with the ester derivatives. Following in the same vein, 18 amide compounds (Table 1) were synthesized ( Figure 3) via reacting the respective phenolic acids with a panel of amines, adopting N,N'-dicyclohexylcarbodiimide (DCC) as a coupling agent.
Therefore, the current study was conducted to verify the anti-tumor activity of CA and FA versus their semisynthetic derivatives on different cell lines and explore the underlying mechanisms using the following scheme:   Therefore, the current study was conducted to verify the anti-tumor activity of CA and FA versus their semisynthetic derivatives on different cell lines and explore the underlying mechanisms using the following scheme:

Chemistry
The synthesis of CA and FA derivatives 3-20 is displayed in Figure 3. Preparation of these candidates was carried out by reacting the naturally obtained caffeic or ferulic acids with different amines using N,N'-dicyclohexylcarbodiimide (DCC) as a coupling agent for the preparation of the amide linkage starting from the acids. Separated amides 3-20 were purified by column chromatography. The main idea is screening the effect of changing the alkyl/cycloalkyl/aralkyl/aryl/heterocyclic moiety of the amide functionality on the biological activity. Compounds 1 and 2 exhibited a sky-blue color in UV, which turns to a greenish yellow color on exposure to ammonia vapor and spraying with AlCl 3, indicating that they are phenolic acid in nature. 1 H NMR spectrum of 1 showed the characteristic signals for caffeic acid [50], which was characterized by two trans-olefinic protons at δ 6.16 and δ 7.41 ppm with a large coupling constant δ 15.88 Hz, assigned to H-8 and H-7, respectively. An ABX system was displayed at δ 6.76, 6.95 and δ 7.04 ppm assigned to H-5, H-6 and H-2, respectively. The 13 C NMR showed the nine characteristic carbon signals for 3,4-dihydroxy trans cinnamic acid with two oxy carbons at δ 146.0 and δ 148.5 (C-3 and 4), one carbonyl carbon at δ 168.3 (C-9), two trans olefinic carbons at δ 145.07 and δ 115.5 ppm, in addition to C-1 at δ 126.1, C-2 at δ 115.1, C-5 at δ 116.2 and C-6 at δ 21.6. 1 H NMR spectrum of 2 showed the characteristic signals for ferulic acid [51], which was characterized by two trans-olefinic protons at δ 6.35 and 7.48 ppm with large coupling constant 15.88 Hz, assigned to H-8 and H-7, respectively. An ABX system was affirmed by these signals at δ 6.79, 7.07 and 7.28 ppm, corresponding to H-5, H-6 and H-2, respectively. The methoxy group at C-3 was manifested by the singlet signal at δ 3.82 ppm and integrated as three protons (Supporting Information).
IR spectra of all the candidates showed the characteristic carbonyl band of amide between 1626-1662 cm −1 while the NH band appeared in the range of 3510-3240 cm −1 for all compounds except 3 and 12, which are tertiary amides. 1 H NMR spectra of compounds 3-20 exhibited the appearance of the doublet signal of HC=CH in the range of δ 5.70-7.73 ppm with large coupling constant 15.00-15.90 Hz. Compounds 3 and 12 revealed the multiplet signals in their 1 H NMR spectra at δ 1.04-1.85 ppm assigned to CH 2 of piperazine ring. 1 H NMR spectra of compounds 5, 14 and 17 showed a series of singlet signals in the range of δ 2.26-2.29 ppm due to CH 3 group while the 11 protons corresponding to the cyclohexyl group in compounds 6 and 15 were confirmed by the appearance of multiplet signals in the range of δ 1-3.35 ppm. Compound 8 was the only CA derivative that showed a singlet signal corresponding to OCH 3 group at δ 3.63 ppm while 16 showed an additional OCH 3 group at δ 3.81 ppm. The appearance of singlet signals at δ 5.59 and 3.19 ppm in 1 H NMR spectra of 9 and 18 respectively confirmed the presence of N-CH 2 (Supporting Information). Compounds 11 and 20 revealed the presence of NH 2 group at δ 9.17 and 4.94 ppm, respectively. 13 C NMR spectrum of all compounds confirmed the presence of the characteristic C=O group, showing signals in the range of δ 160.7-170.9 ppm.

Biological Activity 2.2.1. Cell Viability Determination by MTT Assay
The cytotoxicity of the parent compounds 1 and 2 was tested against breast (MCF-7), liver (HepG2), lung epithelial (A459), colorectal (Caco-2) and pancreatic (PANC-1) cancer cell lines. Their cytotoxicity was compared to doxorubicin (DOX) and their selectivity was tested on Vero cells ( Table 2). The cytotoxicity of both 1 and 2 was lower than that of DOX but the selectivity index (SI) was significantly higher than DOX (Supporting Information). Caco-2 showed the highest sensitivity to both acids 1 and 2 (IC 50 = 0.41 µM for both caffeic and ferulic acids) and thus it was selected for testing the cytotoxicity of the 18 semisynthetic candidates. All the synthesized amides were far more potent than the parent acids by 10-fold or more with a slight reduction in the SI of these derivatives (Table 3). It is worth mentioning that caffeic acid derivatives were more potent than ferulic acid derivatives. Among the CA amide derivatives 3-11, compound 10 was the most potent against the Caco-2 cancer cell line with an IC 50 of 0.009 ± 0.09 µM with a SI 14-fold greater than its IC 50 followed by compounds 11, 7 and 9 with an IC 50 of 0.01 ± 0.11, 0.02 ± 0.11 and 0.03 ± 0.29 µM, respectively. The potency of the rest of the CA amide derivatives ranged from IC 50 = 0.05 µM to 0.08 µM, which was still much more potent than the parent acid. Regarding the FA amide derivatives 12-20, compound 20 revealed the highest potency with an IC 50 of 0.01 ± 0.63 µM followed by compounds 15, 17 and 19 with IC 50 of 0.02 ± 0.17, 0.02 ± 0.21 and 0.02 ± 0.65 µM sequentially. Moreover, compounds 12, 13, 14, 16 and 18 showed very good cytotoxicity against Caco-2 with an IC 50 ranging from 0.04 µM to 0.08 µM. The derivatives were much more potent than the parent acid with an excellent SI (Table 3). To further confirm this, cell-cycle analysis and apoptosis assays were performed and the gene expression of p53, caspase 3, Bax, MMP-2 and MMP-9 was quantified.

Cell Cycle Progression
The potential candidates with anti-tumor effect on the Caco-2 cell line along with a high SI were chosen for cell-cycle analysis. The cell-cycle distribution and cell death were analyzed using Annexin V-FITC/PI staining and flow cytometry (Table 4, Figure 4). The potential candidates that showed potent anticancer activity against the Caco-2 cell line and high SI were chosen to investigate how they will change the cell-cycle distribution. 16.51% respectively) which showed a more significant effect than the parent compound 2 itself (13.22%), whereas the derivatives 17, 18 and 20 showed non-significant decrease in the apoptotic population compared to parent compound 2. (Table 4)

Evaluation of Apoptosis by Annexin V-FITC Staining
Quantification of the different types of apoptotic cells was of our interest due to the accumulation of cells at the G2/M phase during cell-cycle analysis following the treatment with the parent compounds 1 and 2 and their potential candidates.
After Caco-2 cells were treated with parent compounds 1 and 2 and their derivatives,

Flow Cytometry Analysis Showing Induction of Apoptosis
Cell-cycle analysis showed that the treatment of Caco-2 cells with the parent compounds and their derivatives resulted in an increase in the cell population in the G2/M phase, indicating apoptosis. In the case of caffeic acid derivatives, the highest induction of apoptosis was seen in compounds 10, 7 and 11 (23.42%, 18.26% and 14.04%), respectively, which showed a more significant increase than the caffeic acid itself (11.31%). On the contrary, compounds 5 and 9 showed a non-significant increase in the apoptotic population compared to the parent compound 1 (Table 4).
Concerning Ferulic acid derivatives, the accumulation in the Pre-G1 phase and thus the highest induction of apoptosis was observed with candidates 15 and 14 (25.17% and 16.51% respectively) which showed a more significant effect than the parent compound 2 itself (13.22%), whereas the derivatives 17, 18 and 20 showed non-significant decrease in the apoptotic population compared to parent compound 2. (Table 4)

Evaluation of Apoptosis by Annexin V-FITC Staining
Quantification of the different types of apoptotic cells was of our interest due to the accumulation of cells at the G2/M phase during cell-cycle analysis following the treatment with the parent compounds 1 and 2 and their potential candidates.
After Caco-2 cells were treated with parent compounds 1 and 2 and their derivatives, they were stained with annexin V-FITC/PI, and then flow cytometry was used to detect the cell-cycle distribution as shown in Figure 5 and Table 5.    The results of the evaluation of apoptosis revealed that potential candidates 7, 10, 11, 14 and 15 showed the highest proportion of cells in the late apoptotic stage (stained by Annexin V-FITC and PI).

Gene Expression Caffeic Acid and Ferulic Acid Derivatives Enhanced Pro-Apoptotic Genes
The effect of caffeic acid 1 and its amide derivatives 5, 7, 9, 10 and 11 as well as ferulic acid 2 and its derivatives 14, 15, 17, 18, and 20 on the expression of pro-apoptotic genes were tested. β-actin gene was used as a control gene. Gene expression was chosen over Western blotting for more accurate quantification and thus comparison. The gene expression of the p53, caspase 3 and Bax genes was measured. Most of the derivatives showed an increased expression of the three genes, which was evidenced by increasing apoptotic effects. In the case of caffeic acid derivatives, compound 10 showed the highest overexpression with an increase of 14-to 16-fold in the three genes, followed by 7 and 11 ( Figure 6). Regarding the ferulic acid derivatives, 15 showed the highest overexpression of 10-fold in the p53 and 15-fold in the caspase 3 and Bax genes compared to the parent acid ( Figure 7).

Caffeic Acid and Ferulic Acid Derivatives Inhibit Metastasis Enhancing Genes
Matrix metalloproteinases (MMPs) appear to play an important role in the formation and progression of human cancers. MMPs are involved in the breakdown of the extracellular matrix, which is a crucial stage in tumor invasion and spread [52]. A literature review revealed that both 1 and 2 decrease the expression of both MMP-2 and 9, which may lead to decrease in enzymatic activity [53].
The derivatives of both 1 and 2 showed a significant decrease in the expression of both MMP2 and MMP9. Out of the most potential candidates, 17 and 7 showed the greatest inhibition in both groups (Figures 8 and 9).

Structure-Activity Relationship Correlation
The parent phenolic acids 1 and 2 were tested against five cancer cell lines: Caco-2, A549, PANC-1, MCF7, and Hep G2, as well as one normal Vero cell line using DOX as positive reference. The parent compounds showed significant antitumor activity against Caco-2 cell line and with excellent selectivity index (SI). Consequently, all the newly synthesized amide derivatives 3-20 were evaluated for their cytotoxic activity against Caco-2 cancer cell line. The SI of amide derivatives was determined using the Vero normal cell line. They showed excellent cytotoxic activity, much more potent than the parent acids 1 and 2. Based on the phenolic acids scaffold, the amide derivatives were designed to have all the features of both acids as well as the stable amide linkage. All the amide derivatives 3-20 showed very promising anticancer activity against Caco-2 cell line and very good SI. So, changing the carboxylic acid moiety into an amide functionality increased the biological activity and did not destroy the selectivity index of the parent compounds. Examining the structure of the six most active compounds (7, 10, 11, 14, 15 and 17) (Figure 10), starting from the anticancer activity against Caco-2 cell line and excellent SI, followed by cell-cycle analysis and apoptosis assays, then the effect of these candidates on gene expression of p53, caspase 3, Bax, MMP-2 and MMP-9, it was found that besides all the features of both acids and the amide linkage, five compounds out of six have aromatic amide moieties. The presence of p-chloro on the phenyl ring of the amide linkage resulted in the most cytotoxic compound of all the six candidates. Replacing the chloro atom in compound 10 with bromo in compound 7 decreases the antitumor activity. Moreover, it was found that the presence of the cycloalkyl group of the amide linkage in the case of compound 15 increased the induction of apoptosis and showed the highest proportion of cells in late apoptotic stage. In case of enhancing the pro apoptotic genes (p53, caspase 3 and Bax), compound 10 (with p-chlorophenyl) revealed the highest activity. Furthermore, in case of inhibiting the metastasis-enhancing genes, compound 17 with m-tolyl group was the most active one. were tested. β-actin gene was used as a control gene. Gene expression was chosen over Western blotting for more accurate quantification and thus comparison. The gene expression of the p53, caspase 3 and Bax genes was measured. Most of the derivatives showed an increased expression of the three genes, which was evidenced by increasing apoptotic effects. In the case of caffeic acid derivatives, compound 10 showed the highest overexpression with an increase of 14-to 16-fold in the three genes, followed by 7 and 11 ( Figure  6). Regarding the ferulic acid derivatives, 15 showed the highest overexpression of 10-fold in the p53 and 15-fold in the caspase 3 and Bax genes compared to the parent acid ( Figure  7). Figure 6. The effect of CA and its derivatives on the expression of P53, Caspase 3 and Bax genes by RT-qPCR. The results were represented as mean of fold changes ± SD. β-actin gene was used as a control gene. * Significant from CA at p < 0.05, ** Significant from CA at p < 0.01, *** Significant from CA at p < 0.001.

Caffeic Acid and Ferulic Acid Derivatives Inhibit Metastasis Enhancing Genes
Matrix metalloproteinases (MMPs) appear to play an important role in the formation and progression of human cancers. MMPs are involved in the breakdown of the extracellular matrix, which is a crucial stage in tumor invasion and spread [52]. A literature review revealed that both 1 and 2 decrease the expression of both MMP-2 and 9, which may lead to decrease in enzymatic activity [53].
The derivatives of both 1 and 2 showed a significant decrease in the expression of both MMP2 and MMP9. Out of the most potential candidates, 17 and 7 showed the greatest inhibition in both groups (Figures 8 and 9). The results were represented as mean of fold changes ± SD. β-actin gene was used as a control gene. ** Significant from CA at p < 0.01, *** Significant from CA at p < 0.001. The results were represented as mean of fold changes ± SD. β-actin gene was used as a control gene. ** Significant from CA at p < 0.01, *** Significant from CA at p < 0.001. Figure 8. The effect of CA and its derivatives on the expression of MMP-2 and MMP-9 by RT-qPCR. The results were represented as mean of fold changes ± SD. β-actin gene was used as a control gene. ** Significant from CA at p < 0.01, *** Significant from CA at p < 0.001. Figure 9. The effect of FA and its derivatives on the expression of MMP-2 and MMP-9 genes by RT-qPCR. The results were represented as mean of fold changes ± SD. β-actin gene was used as a control gene. *** Significant from FA at p < 0.001. Figure 9. The effect of FA and its derivatives on the expression of MMP-2 and MMP-9 genes by RT-qPCR. The results were represented as mean of fold changes ± SD. β-actin gene was used as a control gene. *** Significant from FA at p < 0.001.

Structure-Activity Relationship Correlation
The parent phenolic acids 1 and 2 were tested against five cancer cell lines: Caco-2, A549, PANC-1, MCF7, and Hep G2, as well as one normal Vero cell line using DOX as positive reference. The parent compounds showed significant antitumor activity against Caco-2 cell line and with excellent selectivity index (SI). Consequently, all the newly synthesized amide derivatives 3-20 were evaluated for their cytotoxic activity against Caco-2 cancer cell line. The SI of amide derivatives was determined using the Vero normal cell line. They showed excellent cytotoxic activity, much more potent than the parent acids 1 and 2. Based on the phenolic acids scaffold, the amide derivatives were designed to have all the features of both acids as well as the stable amide linkage. All the amide derivatives 3-20 showed very promising anticancer activity against Caco-2 cell line and very good SI. So, changing the carboxylic acid moiety into an amide functionality increased the biological activity and did not destroy the selectivity index of the parent compounds. Examining the structure of the six most active compounds (7, 10, 11, 14, 15 and 17) (Figure 10), starting from the anticancer activity against Caco-2 cell line and excellent SI, followed by cell-cycle analysis and apoptosis assays, then the effect of these candidates on gene expression of p53, caspase 3, Bax, MMP-2 and MMP-9, it was found that besides all the features of both acids and the amide linkage, five compounds out of six have aromatic amide moieties. The presence of p-chloro on the phenyl ring of the amide linkage resulted in the most cytotoxic compound of all the six candidates. Replacing the chloro atom in compound 10 with bromo in compound 7 decreases the antitumor activity. Moreover, it was found that the presence of the cycloalkyl group of the amide linkage in the case of compound 15 increased the induction of apoptosis and showed the highest proportion of cells in late apoptotic stage. In case of enhancing the pro apoptotic genes (p53, caspase 3 and Bax), compound 10 (with p-chlorophenyl) revealed the highest activity. Furthermore, in case of inhibiting the metastasis-enhancing genes, compound 17 with m-tolyl group was the most active one.

General
Silica gel 60 (70-230 mesh ASTM; Fluka, Steinheim, Germany), Diaion HP-20 AG, Sephadex LH-20 (Pharmacia Fine Chemicals AB, Uppsala, Sweden) and silica gel GF254 precoated thin layer plates (Fluka, Steinheim, Germany) were used. Chromatograms detections were performed under UV light (at 254 and 366 nm) and sprayed by p-anisaldehyde sulphuric acid spray reagent. Melting points (°C, uncorrected) were determined on Stuart apparatus and the given values were uncorrected. Progress of the reaction was monitored by thin layer chromatography (TLC) using aluminum sheets precoated with UV fluorescent silica gel (MERCK, Rahway, NJ, USA, 60 F 254), and spots were visualized by UV lamp. The solvent system used was dichloromethane: methanol (in different

General
Silica gel 60 (70-230 mesh ASTM; Fluka, Steinheim, Germany), Diaion HP-20 AG, Sephadex LH-20 (Pharmacia Fine Chemicals AB, Uppsala, Sweden) and silica gel GF 254 precoated thin layer plates (Fluka, Steinheim, Germany) were used. Chromatograms detections were performed under UV light (at 254 and 366 nm) and sprayed by p-anisaldehyde sulphuric acid spray reagent. Melting points ( • C, uncorrected) were determined on Stuart apparatus and the given values were uncorrected. Progress of the reaction was monitored by thin layer chromatography (TLC) using aluminum sheets precoated with UV fluorescent silica gel (MERCK, Rahway, NJ, USA, 60 F 254), and spots were visualized by UV lamp. The solvent system used was dichloromethane: methanol (in different ratios). The IR spectra (cm −1 ) were determined using KBr discs on a Shimadzu IR8400s Spectrophotometer (Microanalytical Unit, Faculty of Pharmacy, Cairo University, Cairo, Egypt). 1 H NMR and 13 C NMR spectra were performed on Bruker 400-BB 400 MHz Spectrophotometer, microanalytical unit, Faculty of Pharmacy, Cairo University, Cairo, Egypt, using tetramethylsilane (TMS) as internal standard, and chemical shift values were recorded in ppm on δ scales. Peak multiplicities were designed as follows: s, singlet; d, doublet; t, triplet; m, multiplet. Elemental analyses were performed at the Regional Center for Mycology and Biotechnology, Faculty of Pharmacy, Al-Azhar University, Cairo, Egypt. The data were statistically evaluated using the Graph Pad Prism 6 program. Data were reported as mean ± SD of the triplicates of each experiment. One-way ANOVA with multiple comparisons post-hoc tests were used for analysis. Results were considered significant at p < 0.05.

Plant Material
The grains of oats (Avena sativa L. Family Poaceae) were obtained from the Agricultural Research Center, Giza, Egypt. The plants were authenticated at Botany Department, Faculty of Science, Cairo University, Giza, Egypt. Voucher samples no (3-10-2018) of the plants were deposited at the Museum of the Pharmacognosy Department, Faculty of Pharmacy, Cairo University.

Extraction and Isolation of Caffeic and Ferulic Acids
The powdered oats (2 kg) were extracted with 80% ethanol at 55 • C for 165 min (14 L × three times), the ethanolic extract was evaporated under vacuum at 40 • C. The ethanolic residue (350 gm) was suspended in distilled water (1 L) and defatted by methylene chloride (1 L × 3 times), then the defatted aqueous solution was fractionated over a Dianion column using 100% water, methanol-water (1:1 v/v) and 100% methanol. The solvent in each case was evaporated under vacuum to obtain three fractions 100% water fraction (75 gm), MeOH-water (1:1) fraction (55 gm) and 100% methanol fraction 67 gm.

Annexin V/Propidium Iodide (PI) Apoptosis Assay by Flow Cytometry
In order to determine the percentage of viable, apoptotic and necrotic cells after treatment with our compounds, double-staining with fluorescein isothiocyanate (FITC) and PI was performed. Annexin V-FITC Apoptosis Detection Kit (BioVision, CA, USA) was used. Briefly, cultured Caco-2 were treated with the tested compounds or DMSO for 24h. Cells were then collected by centrifugation at 2000 rpm for 5 min, and the pellet was washed with PBS then centrifuged again at 2000 rpm for 5 min then resuspended in 500µL of the kit's binding buffer before adding the Annexin V-FITC and PI. The cells were then incubated at room temperature in the dark for 5 min. Annexin V-FITC binding was measured using FACS Calibur flow cytometer (BD biosciences, USA) (Ex = 488 nm, Em = 533 nm, FL1 filter for annexin and FL2 filter for PI).

Quantitative Polymerase Chain Reaction (qPCR) and RNA Extraction
The treated cells were harvested and the total RNA was extracted using RNeasy Mini Kit (Qiagen) according to the manufacturer's instruction. The primers of the required genes (Table 6) were used for SYBR Green qRT-PCR using a one-step RT-PCR kit with SYBR Green (Bio-Rad). qRT-PCR was performed on Rotor Gene Q (Qiagen). The mRNA levels of p53, Bcl-2, Caspase-3, Bax, MMP-2, and MMP-9 were calculated using the 2 −∆∆CT method with the endogenous β-actin mRNA as control to normalize the values. Rotor-Gene 6000 Series Software was used for data analysis. and their HGNC ID was added ( Table 6) Table 6. Primers used in qRT-PCR.

Conclusions
Despite the high efficacy of CA and FA, they are limited by their high metabolism and consequent low bioavailability. Therefore, we were challenged to synthesize highly stable and biologically active compounds. Henceforth, we designed and investigated 18 congeners of amide nature; they were all superior to their parent acids in terms of cytotoxic activity against Caco-2. The selected candidates for cell-cycle analysis and gene expression assays were those displaying superior cytotoxicity and high SI using Vero normal cells.
Subsequently, the assays offered six remarkably active compounds, 7, 10, 11, 14, 15 and 17, and five of them comprised an aromatic ring adjacent to the amide bridge. In accordance with the SAR, the highest cytotoxicity was evident amongst the aromatic derivative with p-chloro phenyl 10, displaying excellent cytotoxic activity, this was deduced when the cytotoxicity decreased after the chloro residue was replaced with a bromo substitution. It could also be concluded that overall, the caffeic acid amides surpassed the ferulic analogues.
We are planning to further study other amide derivatives and assess their activity in a suitable animal model.